Imidazole-2-carboxamide derivatives as Raf kinase inhibitors

ABSTRACT

The present invention relates to compounds of formula (I): 
                         
or pharmaceutically acceptable salts thereof, corresponding pharmaceutical compositions, use as Raf Kinase Inhibitors and treatment methods for neurotraumatic diseases and cancer.

This invention relates to novel compounds and their use aspharmaceuticals particularly as Raf kinase inhibitors for the treatmentof neurotraumatic diseases.

Raf protein kinases are key components of signal transduction pathwaysby which specific extracellular stimuli elicit precise cellularresponses in mammalian cells. Activated cell surface receptors activateras/rap proteins at the inner aspect of the plasmamembrane which in turnrecruit and activate Raf proteins. Activated Raf proteins phosphorylateand activate the intracellular protein kinases MEK1 and MEK2. In turn,activated MEKs catalyse phosphorylation and activation of p42/p44mitogen-activated protein kinase (MAPK). A variety of cytoplasmic andnuclear substrates of activated MAPK are known which directly orindirectly contribute to the cellular response to environmental change.Three distinct genes have been identified in mammals that encode Rafproteins; A-Raf, B-Raf and C-Raf (also known as Raf-1) and isoformicvariants that result from differential splicing of mRNA are known.

Inhibitors of Raf kinases have been suggested for use in disruption oftumor cell growth and hence in the treatment of cancers, e.g.histiocytic lymphoma, lung adenocarcinoma, small cell lung cancer andpancreatic and breast carcinoma; and also in the treatment and/orprophylaxis of disorders associated with neuronal degeneration resultingfrom ischemic events, including cerebral ischemia after cardiac arrest,stroke and multi-infarct dementia and also after cerebral ischemicevents such as those resulting from head injury, surgery and/or duringchildbirth.

We have now found a group of novel compounds that are inhibitors of Rafkinases, in particular inhibitors of B-Raf kinase.

According to the invention there is provided a compound of formula (I):

wherein

X is O, CH₂, S or NH, or the moiety X—R¹ is hydrogen;

Y₁ and Y₂ independently represent CH or N;

R¹ is hydrogen, C₁₋₆alkyl, C₃₋₇cycloalkyl, aryl, arylC₁₋₆alkyl,heterocyclyl, heterocyclylC₁₋₆alkyl, heteroaryl, or heteroarylC₁₋₆alkylany of which except hydrogen, may be optionally substituted;

R² and R³ independently represent hydrogen, C₁₋₆alkyl, C₃₋₇cycloalkyl,aryl, arylC₁₋₆alkyl, heteroaryl, heteroarylC₁₋₆alkyl, heterocyclyl, orheterocyclylC₁₋₆alkyl, or R² and R³ together with the nitrogen atom towhich they are attached form a 4- to 10-membered monocyclic or bicyclicring;

Ar is a group of the formula a) or b):

wherein A represents a fused 5- to 7-membered ring optionally containingup to two heteroatoms selected from O, S and NR⁵, wherein R⁵ is hydrogenor C₁₋₆alkyl, which ring is optionally substituted by up to 2substituents selected from halogen, C₁₋₆alkyl, hydroxy, C₁₋₆alkoxy orketo;

R^(a) and R⁴ are independently selected from hydrogen, halogen,C₁₋₆alkyl, aryl, arylC₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl,haloC₁₋₆alkyl, arylC₁₋₆alkoxy, hydroxy, nitro, cyano, azido, amino,mono- and di-N—C₁₋₆alkylamino, acylamino, arylcarbonylamino, acyloxy,carboxy, carboxy salts, carboxy esters, carbamoyl, mono- anddi-N—C₁₋₆alkylcarbamoyl, C₁₋₆alkoxycarbonyl, aryloxycarbonyl, ureido,guanidino, C₁₋₆alkylguanidino, amidino, C₁₋₆alkylamidino,sulphonylamino, aminosulphonyl, C₁₋₆alkylthio, C₁₋₆alkyl sulphinyl orC₁₋₆alkylsulphonyl; and

one of X₁ and X₂ is N and the other is NR⁶, wherein R⁶ is hydrogen,C₁₋₆alkyl, or arylC₁₋₆alkyl;

or a pharmaceutically acceptable salt thereof.

As used herein, the double bond indicated by the dotted lines of formula(I), represent the possible tautomeric ring forms of the compoundsfalling within the scope of this invention, the double bond being to theunsubstituted nitrogen atom.

The hydroxyimino moiety can be positioned on any of carbon atoms of thenon-aromatic ring in groups a) and b).

The hydroxyimino moiety can exist as either the E or Z isomer or as amixture of both.

Alkyl and alkenyl groups referred to herein, individually or as part oflarger groups e.g. alkoxy, may be straight or branched groups containingup to six carbon atoms and are optionally substituted by one or moregroups selected from the group consisting of aryl, heteroaryl,heterocyclyl, C₁₋₆alkoxy, C₁₋₆alkylthio, arylC₁₋₆alkoxy,arylC₁₋₆alkylthio, amino, mono- or di-C₁₋₆alkylamino, cycloalkyl,cycloalkenyl, carboxy and esters thereof, amide, sulphonamido, ureido,guanidino, C₁₋₆alkylguanidino, amidino, C₁₋₆alkylamidino, C₁₋₆ acyloxy,azido, hydroxy, hydroxyimino and halogen.

Cycloalkyl and cycloalkenyl groups referred to herein include groupshaving from three to seven ring carbon atoms and are optionallysubstituted as described hereinabove for alkyl and alkenyl groups.

When used herein, the term “aryl” includes, unless otherwise defined,single and fused rings suitably containing from 4 to 7, preferably 5 or6, ring atoms in each ring, which rings, may each be unsubstituted orsubstituted by, for example, up to three substituents.

Suitable aryl groups include phenyl and naphthyl such as 1-naphthyl or2-naphthyl.

Optional substituents for alkyl, alkenyl, cycloalkyl and cycloalkenylgroups include aryl, heteroaryl, heterocyclyl, C₁₋₆alkoxy,C₁₋₆alkylthio, arylC₆alkoxy, arylC₁₋₆alkylthio, amino, mono-ordi-C₁₋₆alkylamino, aminosulphonyl, cycloalkyl, cycloalkenyl, carboxy andesters thereof, amide, ureido, quanidino, C₁₋₆alkylquanidino, amidino,C₁₋₆alkylamidino, C₁₋₆acyloxy, hydroxy, and halogen or any combinationthereof.

When used herein the term “heterocyclyl” includes, unless otherwisedefined, non-aromatic, single and fused, rings suitably containing up tofour heteroatoms in each ring, each of which is selected from O, N andS, which rings, may be unsubstituted or substituted by, for example, upto three substituents. Each heterocyclic ring suitably has from 4 to 7,preferably 5 or 6, ring atoms. A fused heterocyclic ring system mayinclude carbocyclic rings and need include only one heterocyclic ring.Examples of heterocyclyl groups include pyrrolidine, piperidine,piperazine, morpholine, imidazolidine and pyrazolidine.

When used herein, the term “heteroaryl” includes, unless otherwisedefined, mono- and bicyclic heteroaromatic ring systems comprising up tofour, preferably 1 or 2, heteroatoms each selected from O, N and S. Eachring may have from 4 to 7, preferably 5 or 6, ring atoms. A bicyclicheteroaromatic ring system may include a carbocyclic ring. Examples ofheteroaryl groups include pyrrole, quinoline, isoquinoline, pyridine,pyrimidine, oxazole, thiazole, thiadiazole, triazole, imidazole andbenzimidazole.

Aryl, heterocyclyl and heteroaryl groups may be optionally substitutedby preferably up to three substituents. Suitable substituents includehalogen, C₁₋₆alkyl, aryl, aryl C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl, halo C₁₋₆alkyl, arylC₁₋₆alkoxy, hydroxy, nitro, cyano, azido,amino, mono- and di-N—C₁₋₆alkylamino, acylamino, arylcarbonylamino,acyloxy, carboxy, carboxy salts, carboxy esters, carbamoyl, mono- anddi-N—C₁₋₆alkylcarbamoyl, C₁₋₆alkoxycarbonyl, aryloxycarbonyl, ureido,guanidino, C₁₋₆alkylguanidino, amidino, C₁₋₆alkylamidino,sulphonylamino, aminosulphonyl, C₁₋₆alkylthio, C₁₋₆alkylsulphinyl,C₁₋₆alkylsulphonyl, heterocyclyl, heteroaryl, heterocyclyl C₁₋₆alkyl,hydroxyimino-C₁₋₆alkyl and heteroaryl C₁₋₆alkyl, and combinationsthereof.

Preferably the optional substituent contains a solubilising group;suitable solubilising moieties will be apparent to those skilled in theart and include hydroxy and amine groups. Even more preferably theoptional substituent includes amino, mono- or di-C₁₋₆alkylamino, aminecontaining heterocyclyl, and hydroxy or any combination thereof.

X is preferably NH or X—R¹ is preferably hydrogen and when X is NH, R¹is preferably hydrogen or C₁₋₆alkyl.

When Y₁ and Y₂ are CH, X—R¹ is preferably hydrogen.

When Y₂ is N, X—R¹ is preferably NH₂.

Preferrably R⁶ is hydrogen.

Most preferably X—R¹ is hydrogen

A is preferrably a fused 5 membered ring optionally containing up to twoheteroatoms selected from O, S and NR⁵, wherein R⁵ is hydrogen orC₁₋₆alkyl, which ring is optionally substituted by up to 2 substituentsselected from halogen, C₁₋₆alkyl, hydroxy, C₁₋₆alkoxy or keto.

Even more preferably A is a a fused 5 membered ring.

Preferably R² and R³ independently represent hydrogen, C₁₋₆alkyl, aryl,heteroarylC₁₋₆alkyl, heterocyclyl, heterocyclylC₁₋₆alkyl, any of whichexcept hydrogen can be optionally substituted or R² and R³ together withthe nitrogen atom to which they are attatched form an optionallysubstituted 5 or 6 membered monocyclic or bicyclic ring for examplepiperidine.

The compounds of formula (I) preferably have a molecular weight of lessthan 800.

Preferred substituents for the group Ar include halo, hydroxy, hydroxyC₁₋₆alkyl, hydroxyimino-C₁₋₆alkyl and C₁₋₆alkoxy.

It will be understood that the invention includes pharmaceuticallyacceptable derivatives of compounds of formula (I) and that these areincluded within the scope of the invention.

Particular compounds according to the invention include those mentionedin the examples and their pharmaceutically acceptable salts. As usedherein “pharmaceutically acceptable derivatives” includes anypharmaceutically acceptable salt, ester or salt of such ester of acompound of formula (I) which, upon administration to the recipient, iscapable of providing (directly or indirectly) a compound of formula (I)or an active metabolite or residue thereof.

It will be appreciated that for use in medicine the salts of thecompounds of formula (I) should be pharmaceutically acceptable. Suitablepharmaceutically acceptable salts will be apparent to those skilled inthe art and include those described in J. Pharm. Sci., 1977, 66, 1-19,such as acid addition salts formed with inorganic acids e.g.hydrochloric, hydrobromic, sulfuric, nitric or phosphoric acid; andorganic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric,benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid.Other salts e.g. oxalates, may be used, for example in the isolation ofcompounds of formula (I) and are included within the scope of thisinvention.

The compounds of this invention may be in crystalline or non-crystallineform, and, if crystalline, may optionally be hydrated or solvated. Thisinvention includes within its scope stoichiometric hydrates as well ascompounds containing variable amounts of water.

The invention extends to all isomeric forms including stereoisomers andgeometric isomers of the compounds of formula (I) including enantiomersand mixtures thereof e.g. racemates. The different isomeric forms may beseparated or resolved one from the other by conventional methods, or anygiven isomer may be obtained by conventional synthetic methods or bystereospecific or asymmetric syntheses.

Since the compounds of formula (I) are intended for use inpharmaceutical compositions it will readily be understood that they areeach preferably provided in substantially pure form, for example atleast 60% pure, more suitably at least 75% pure and preferably at least85%, especially at least 98% pure (% are on a weight for weight basis).Impure preparations of the compounds may be used for preparing the morepure forms used in the pharmaceutical compositions.

Compounds of Formula (I) are imidazole derivatives which may be readilyprepared using procedures well-known to those skilled in the art, anddescribed in, for instance, Comprehensive Heterocyclic Chemistry,Editors Katritzky and Rees, Pergamon Press, 1984, 5, 457-497, fromstarting materials which are either commercially available or can beprepared from such by analogy with well-known processes. A key step inmany such syntheses is the formation of the central imidazole nucleus.

Preferred methods for preparing compounds of this invention are asoutlined in the above scheme. α-Diketones are prepared by condensationof the anion of, for example, a 4-substituted pyridine derivative(Y₁═Y₂═CH, R¹—X═H and R⁴═R^(a)═H) with a suitably protected fusedbicyclic arylaldehyde (e.g. 1-methoxyimino-indan-5-carboxaldehyde)followed by oxidation of the intermediate product. Stirring the diketonewith an aldehyde, such as glyoxylic acid ethyl ester, and ammoniumacetate in a mixture of methanol and methyl-tert-butyl ether allowsaccess to the imidazole nucleus, by analogy to the method described inpatent WO 98/56788. Thereafter, the ethyl ester may be converted into anamide using conventional amide bond forming procedures (such proceduresare well known in the art and are described in, for instance, P. D.Bailey, I. D. Collier and K. M. Morgan in Comprehensive OrganicFunctional Group Transformation, Vol. 5, ed. C. J. Moody, p.257,Elsevier Scientific, Oxford, 1995.) and the group PG converted into anhydroxyimono group (HO—N═).

Non-selective alkylation of the imidazole nitrogen (using one of theprocedures outlined in N. J. Liverton et al; J. Med. Chem., 1999, 42,2180-2190) with a compound of formula L-R⁴ wherein L is a leaving group,e.g. halo, sulfonate or triflate, will yield both isomers of thecompounds where X₁ or X₂ is NR⁶ in which R⁶ is other than hydrogen, theisomers can be separated by chromatographic methods.

During the synthesis of the compounds of formula (I) labile functionalgroups in the intermediate compounds, e.g. hydroxy, carboxy and aminogroups, may be protected. A comprehensive discussion of the ways inwhich various labile functional groups groups may be protected andmethods for cleaving the resulting protected derivatives is given in forexample Protective Groups in Organic Chemistry, T. W. Greene and P. G.M. Wuts, (Wiley-Interscience, New York, 2nd edition, 1991).

The compounds of formula (I) may be prepared singly or as compoundlibraries comprising at least 2, for example 5 to 1,000 compounds, andmore preferably 10 to 100 compounds of formula (I). Libraries ofcompounds of formula (I) may be prepared by a combinatorial ‘split andmix’ approach or by multiple parallel synthesis using either solutionphase or solid phase chemistry, by procedures known to those skilled inthe art.

Thus according to a further aspect of the invention there is provided acompound library comprising at least 2 compounds of formula (I), orpharmaceutically acceptable salts thereof.

Pharmaceutically acceptable salts may be prepared conventionally byreaction with the appropriate acid or acid derivative.

The novel carboxylic esters and the corresponding acids of formula (II)and (III) which are used as intermediates in the synthesis of thecompounds of formula (I) also form part of the present invention:

wherein X, Y₁, Y₂, R¹, R^(a), Ar, X₁ and X₂ are as defined for formula(I) and R is hydrogen, C₁₋₆alkyl or arylC₁₋₆alkyl.

As indicated above the compounds of formula (I) and theirpharmaceutically acceptable derivatives are useful for the treatmentand/or prophylaxis of disorders in which Raf kinases, in particularB-Raf kinase, are implicated.

According to a further aspect of the invention there is provided the useof a compound of formula (I) or a pharmaceutically acceptable saltthereof as an inhibitor of B-Raf kinase.

As indicated above the compounds of formula (I) and theirpharmaceutically acceptable derivatives are useful the treatment and/orprophylaxis of disorders associated with neuronal degeneration resultingfrom ischemic events.

According to a futher aspect of the invention there is provided a methodof treatment or prophylaxis of a neurotraumatic disease, in a mammal inneed thereof, which comprises administering to said mammal an effectiveamount of a compound of formula (I) or a pharmaceutically acceptablederivative thereof.

According to a futher aspect of the invention there is provided the useof a compound of formula (I) or a pharmaceutically acceptable derivativethereof in the manufacture of a medicament for the prophylactic ortherapeutic treatment of any disease state in a human, or other mammal,which is exacerbated or caused by a neurotraumatic event.

Neurotraumatic diseases/events as defined herein include both open orpenetrating head trauma, such as caused by surgery, or a closed headtrauma injury, such as caused by an injury to the head region. Alsoincluded within this definition is ischemic stroke, particularly to thebrain area, transient ischemic attacks following coronary by-pass andcognitive decline following other transient ischemic conditions.

Ischemic stroke may be defined as a focal neurologic disorder thatresults from insufficient blood supply to a particular brain area,usually as a consequence of an embolus, thrombi, or local atheromatousclosure of the blood vessel. Roles for stress stimuli (such as anoxia),redox injury, excessive neuronal excitatory stimulation and inflammatorycytokines in this area has been emerging and the present inventionprovides a means for the potential treatment of these injuries.Relatively little treatment, for an acute injury such as these has beenavailable.

The compounds of the invention may also be used in the treatment orprophylaxis of cancers.

In order to use the compounds of formula (I) in therapy, they willnormally be formulated into a pharmaceutical composition in accordancewith standard pharmaceutical practice.

According to a futher aspect of the invention there is provided apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable derivative thereof and a pharmaceuticallyacceptable carrier.

The compounds of formula (I) may conveniently be administered by any ofthe routes conventionally used for drug administration, for instance,parenterally, orally, topically or by inhalation. The compounds offormula (I) may be administered in conventional dosage forms prepared bycombining it with standard pharmaceutical carriers according toconventional procedures. The compounds of formula (I) may also beadministered in conventional dosages in combination with a known, secondtherapeutically active compound. These procedures may involve mixing,granulating and compressing or dissolving the ingredients as appropriateto the desired preparation. It will be appreciated that the form andcharacter of the pharmaceutically acceptable carrier is dictated by theamount of compound of formula (I) with which it is to be combined, theroute of administration and other well-known variables. The carrier(s)must be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipientthereof.

The pharmaceutical carrier employed may be, for example, either a solidor liquid. Exemplary of solid carriers are lactose, terra alba, sucrose,talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acidand the like. Exemplary of liquid carriers are syrup, peanut oil, oliveoil, water and the like. Similarly, the carrier or diluent may includetime delay material well known to the art, such as glycerylmono-stearate or glyceryl distearate alone or with a wax.

A wide variety of pharmaceutical forms can be employed. Thus, if a solidcarrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg to about 1 g. When a liquid carrier is used,the preparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable liquid such as an ampoule or nonaqueousliquid suspension.

The compounds of formula (I) are preferably administered parenterally,that is by intravenous, intramuscular, subcutaneous intranasal,intrarectal, intravaginal or intraperitoneal administration. Theintravenous form of parenteral administration is generally preferred.The compounds may be administered as a bolus or continuous infusion e.g.over 3 days. Appropriate dosage forms for such administration may beprepared by conventional techniques.

The compounds of formula (I) may also be administered orally.Appropriate dosage forms for such administration may be prepared byconventional techniques.

The compounds of formula (I) may also be administered by inhalation,that is by intranasal and oral inhalation administration. Appropriatedosage forms for such administration, such as aerosol formulations, maybe prepared by conventional techniques.

The compounds of formula (I) may also be administered topically, that isby non-systemic administration. This includes the application of theinhibitors externally to the epidermis or the buccal cavity and theinstillation of such a compound into the ear, eye and nose, such thatthe compound does not significantly enter the blood stream.

For all methods of use disclosed herein the daily oral dosage regimenwill preferably be from about 0.1 to about 80 mg/kg of total bodyweight, preferably from about 0.2 to 30 mg/kg, more preferably fromabout 0.5 mg to 15 mg. The daily parenteral dosage regimen about 0.1 toabout 80 mg/kg of total body weight, preferably from about 0.2 to about30 mg/kg, and more preferably from about 0.5 mg to 15 mg/kg. The dailytopical dosage regimen will preferably be from 0.1 mg to 150 mg,administered one to four, preferably two or three times daily. The dailyinhalation dosage regimen will preferably be from about 0.01 mg/kg toabout 1 mg/kg per day. It will also be recognized by one of skill in theart that the optimal quantity and spacing of individual dosages of theinhibitors will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration, and theparticular patient being treated, and that such optimums can bedetermined by conventional techniques. It will also be appreciated byone of skill in the art that the optimal course of treatment, i.e., thenumber of doses of the inhibitors given per day for a defined number ofdays, can be ascertained by those skilled in the art using conventionalcourse of treatment determination tests. In the case of pharmaceuticallyacceptable salts the above figures are calculated as the parent compoundof formula (I).

No toxicological effects are indicated/expected when a compound offormula (I) is administered in the above mentioned dosage range.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

The following Examples illustrate the preparation of pharmacologicallyactive compounds of the invention and the following Descriptionsillustrate the preparation of intermediates used in the preparation ofthese compounds.

Abbreviations uesd herein are as follows;

-   TBF means tetrahydrofuran.-   DMF means N,N-Dimethylformamide.-   LDA means lithium diisopropylamide.-   TBAF means tetrabutylammonium fluoride.-   DMSO means methyl sulfoxide.

DESCRIPTION 1:4-(1-Oxo-indan-5-yl)-5-pyridin-4-yl-1H-imidazole-2-carboxylic Acid

Step 1. 5-Bromo-indan-1-one O-methyl-oxime

To a solution of 5-bromo-indanone (100 g, 0.474 mol) in ethanol (650 ml)under argon was added methoxylamine hydrochloride (198 g, 2.38 mol) andpyridine (125 ml). The mixture was refluxed for 2.5 hours, cooled toroom temperature and poured into saturated aqueous sodium hydrogencarbonate solution. The mixture was then extracted with ethyl acetateand the organic phase dried (Na₂SO₄) and then concentrated in vacuo. Thecrude material was recrystallised from isopropanol to furnish the titlecompound, (110 g, 97%), as a brown solid; ¹H NMR (CDCl₃) 7.52 (1H, d, J8.3 Hz), 7.43 (1H, d, J 1 Hz), 7.35 (1H, dd, J 8.3, 1 Hz), 3.97 (3H, s),2.99 (2H, m), 2.85 (2H, m).

Step 2. 1-Methoxyimino-indan-5-carbaldehyde

To a solution of the product of Step 1 (112 g, 0.46 mol) in THF (1500ml) at −60° C. under argon, was added n-BuLi (325 ml, 0.52 mol) over 1hour. After stirring at −60° C. for 1 hour a solution of DMF (39.7 ml)in THF (50 ml) was added dropwise over 1 hour. The reaction was stirredat −60° C. for 1 hour before being allowed to warm to room temperature.After 1 hour the reaction was quenched with saturated aqueous sodiumhydrogen carbonate solution and extracted into ethyl acetate. Theorganic phase was then dried (Na₂SO₄), concentrated in vacuo and theresidue purified by silica gel chromatography, to give the titlecompound (57 g, 65%) as a yellow solid; ¹H NMR (CDCl₃) 10.0 (1H, s),7.83-7.73 (3H, m), 4.02 (3H, s), 3.10 (2H, m), 2.92 (2H, m).

Step 3.5-(1,2-Dihydroxy-2-pyridin-4-yl-ethyl)-indan-1-one-O-methyl-oxime

To a solution of 4-(tert-butyl-dimethyl-silyloxymethyl)-pyridine [T. F.Gallagher et al, Bioorg. Med. Chem., 1997, 5, 49] (71.5 g, 0.32 mol) inTHF (800 ml) at −50° C. under argon was added LDA (162 ml, 2M inheptane/THF/ethylbenzene, 0.324 mol) over 1 hour. The mixture wasstirred at −40° C. for a further 1 hour before a solution of the productof Step 2 (55 g, 0.29 mol) in THF (600 ml) was added over 1 hour. Thereaction was then allowed to warm to room temperature overnight beforebeing quenched by the addition of saturated aqueous sodium hydrogencarbonate solution and then extracted into ethyl acetate. The organicphase was dried (Na₂SO₄) and concentrated in vacuo to give a brown oil(125 g).

The oil was then dissolved in THF (1500 ml), treated with TBAF (356 ml,0.356 mol) and stirred for 1 hour. The reaction mixture was thenevaporated and the residue partitioned between water and ethyl acetate.The organic phase was then dried (Na₂SO₄) and concentrated to give thetitle compound (57 g, 64%) as a pale yellow solid which was used withoutfurther purification. ¹H NMR (CDCl₃) 8.38 (2H, m), 7.57 (1H, m),7.12-6.99 (4H, m), 4.88 (1H, m), 4.66 (1H, m), 3.96 (3H, s), 2.93 (2H,m), 2.85 (2H, m).

Step 4. 1-(1-Methoxyimino-indan-5-yl)₂-pyridin-4-yl-ethane-1,2-dione

To a mixture of DMSO (43 ml, 0.56 mol) and dichloromethane (800 ml) at−70° C. under argon, was added oxalyl chloride (43.2 g) and then asolution of the product of Step 3 (55 g, 0.185 mol) in a mixture ofdichloromethane/DMSO (1000 ml/60 ml) over 2 hours at −60° C. Afterstirring for 2 hours at −60° C., triethylamine (154 ml) was addeddropwise and the mixture then allowed to warm to room temperatureovernight. The reaction mixture was then quenched with water, theorganic phase separated then washed with water, dried (Na₂SO₄) andconcentrated to yield the title compound (51 g, 94%) as a yellow solid.¹H NMR (CDCl₃) 8.87 (2H, d), 7.89-7.77 (5H, m), 4.03 (3H, s), 3.09(2H,m), 2.93 (2H, m).

Step 5.4-(1-Methoxyimino-indan-5-yl)-5-pyridin-4yl-1H-imidazole-2-carboxylicAcid Ethyl Ester

A mixture of the product of Step 4 (4.3 g, 15 mmol) and glyoxylic acidethyl ester (50% solution in toluene, 6 ml, 30 mmol) intert-butyl-methyl ether (150 ml) at 5° C. was treated with a solution ofammonium acetate (11.2 g, 145 mmol) in methanol (50 ml). After stirringat room temperature for 2 hours the solution was evaporated in vacuo andthe residue was partitioned between chloroform and saturated sodiumhydrogen carbonate solution. The organic layer was separated, washedwith water and brine, dried MgSO₄), filtered and concentrated at reducedpressure. Purification of the residue by chromatography on silica geleluting with 5% methanol in chloroform gave the title compound as ayellow solid (1.0 g, 18%); MS(AP+) m/e 377 [M+H]⁺.

Step 6. 4-(1-Oxo-indan-5-yl)-5-pyridin-4-yl-1H-imidazole-2-carboxylicAcid Ethyl Ester Dihydrochloride Salt

A mixture of the product of Step 5 (1.0 g, 2.7 mmol) and 5M HCl (15 ml)in dioxane (30 ml)/acetone (30 ml) was heated to 100° C. for 3 hour. Themixture was cooled to room temperature and the solvent evaporated invacuo. The residue was co-evaporated with acetone (2×20 ml) andethanol/acetone (1:1, 20 ml) to give the title compound (1.0 g, 88%);MS(ES⁻) m/e 346 [M−H]⁻.

Step 7. 4-(1-Oxo-indan-5-yl)-5-pyridin-4-yl-1H-imidazole-2-carboxylicAcid

A solution of the product of Step 6 (1.0 g, 2.4 mmol) in ethanol (10 ml)containing, 10% aqueous sodium hydroxide solution (10 ml) was heated at50° C. overnight. On cooling, the solvent was removed in vacuo, theresidue dissolved in water and the pH adjusted to 6 with glacial aceticacid and the solvent re-evaporated. The residue was suspended in water(50 ml) and the solid was collected, washed with water and dried overphosphorous pentoxide to give the crude title compound as an off-whitesolid (0.7 g); MS(AP⁻) m/e 318 [M−H]⁻.

EXAMPLE 14-(1-Hydroxyimino-indan-5-yl)-5-pyridin-4-yl-1H-imidazole-2-carboxylicAcid (2-morpholin-4-yl-ethyl)-amide

Step 1. 4-(1-Oxo-indan-5-yl)-5-pyridin-4-yl-1H-imidazole-2-carboxylicAcid (2-morpholin-4-yl-ethyl)-amide

A mixture of the product of Description 1 (105 mg, 0.33 mmol),1-hydroxybenzotriazole (68 mg, 0.5 mmol) andN-cyclohexylcarbodiimide-N′-methyl polystyrene (500 mg, 0.66 mmol, resinloading 1.32 mmol/g) in DMF (2 ml) was stirred at room temperature for30 minutes. A solution of 2-morpholin-4-yl-ethylamine (43 mg, 0.33 mmol)in dichloromethane (0.5 ml) was added and the mixture stirred at roomtemperature for 24 hours. The mixture was filtered through an SCX cationexchange resin column eluting with methanol followed by 10% 0.880ammonia solution in methanol to elute the product. Followingconcentration, the residue was purified by silica gel chromatographyeluting with a 1:9:90 mixture of 0.880 ammonia solution:methanol:chloroform gave the title compound (65 mg, 46%) as a yellowsolid; MS(AP+) m/e 432 [M+H]⁺.

Step 2.4-(1-Hydroxyimino-indan-5-yl)-5-pyridin-4-yl-1H-imidazole-2-carboxylicAcid (2-morpholinyl-ethyl)-amide

A solution of the product of Step 1 (65 mg, 0.15 mmol) in ethanol (4 ml)and aqueous hydroxylamine (1 ml, 50% in water) was heated under refluxfor 1 hour. After cooling to room temperature, the solution wasconcentrated in vacuo and the residue co-evaporated with ethanol (3×10ml). Purification of the residue by silica gel chromatography elutingwith a 1:9:90 mixture of 0.88 ammonia solution: methanol:chloroform gavethe title compound (50 mg, 75%) as a yellow solid; MS(AP+) m/e 447[M+H]⁺.

The following examples were prepared from by the general two-step methoddescribed in Example 1.

Example Amine Characterisation 2 5-(2-{1-[4-(2- 1-(2-Methoxy- MS(AP+)m/e Methoxy-ethyl)-piperazin- ethyl)-piperazine 461 [M + H]⁺1-yl]-methanoyl}- 5-pyridin-4-yl-1H- imidazol-4-yl)-indan-1- one oxime 34-(1-Hydroxyimino- Methyl-(1- MS(AP+) m/e indan-5-yl)-5-methyl-piperidin- 445 [M + H]⁺ pyridin-4-yl-1H- 4-yl)-amineimidazol-2-carboxylic acid methyl-(1-methyl- piperidin-4-yl)-amide 45-{2-[1-(4- 1-Cyclohexyl- MS(AP+) m/e Cyclohexyl-piperazin- piperazine485 [M + H]⁺ 1-yl)-methanoyl]-5- pyridin-4-yl-1H-imidazol-4-yl}-indan-1-one oxime 5 5-[2-(1-[1,4′]- 4-Piperidino- MS(AP+) m/eBipiperidinyl-1′-yl- piperidine 485 [M + H]⁺ methanoyl)-5-pyridin-4-yl-1H-imidazol-4- yl]-indan-1-one oxime 6 5-(2-{1-[4-(4-Chloro-1-(4-Chloro- MS(AP+) m/e benzyl)-piperazin-1- benzyl)- 453 [M + H]⁺yl]-methanoyl}-5- piperazine pyridin-4-yl-1H-imidazol- 4-yl)-indan-1-oneoxime 7 4-(1-Hydroxyimino-indan- 3- MS(AP+) m/e 5-yl)-5-pyridin-4-yl-Dimethylamino- 467 [M + H]⁺ 1H-imidazol-2-carboxylic methyl- acid (3-phenylamine dimethylaminomethyl- phenyl)-amide 8 4-(1-Hydroxyimino-[4-(2- MS(AP+) m/e indan-5-yl)-5-pyridin- Diisopropyl- 597 [M + H]⁺4-yl-1H-imidazol-2- amino-ethoxy)-3- carboxylic acid [4-methoxy-phenyl]- (2-diisopropylamino- methyl-amine ethoxy)-3-methoxy-phenyl]-methyl-amide

It is to be understood that the present invention covers allcombinations of particular and preferred subgroups describedhereinabove.

BIOLOGICAL EXAMPLES

The activity of compounds of formula (I) as B-Raf inhibitors may bedetermined by the following in vitro assay:

Fluorescence Anisoptrophy Kinase Binding Assay

The kinase enzyme, fluorescent ligand and a variable concentration oftest compound are incubated together to reach thermodynamic equilibriumunder conditions such that in the absence of test compound thefluorescent ligand is significantly (>50%) enzyme bound and in thepresence of a sufficient concentration (>10×Ki) of a potent inhibitorthe anisotropy of the unbound fluorescent ligand is measurably differentfrom the bound value.

The concentration of kinase enzyme should preferably be≧1×K_(f). Theconcentration of fluorescent ligand required will depend on theinstrumentation used, and the fluorescent and physicochemicalproperties. The concentration used must be lower than the concentrationof kinase enzyme, and preferably less than half the kinase enzymeconcentration. A typical protocol is:

All compounds dissolved in Buffer of comparison 50 mM HEPES,pharmaceutical 7.5, 1 mM CHAPS, 10 mM MgCL₂.

B-Raf Enzyme concentration: 1 nM

Fluorescent ligand concentration: 0.5 nM

Test compound concentration: 0.5 nM-100 uM

Components incubated in 10 ul final volume in LJL HE 384 type B blackmicrotitre plate until equilibrium reached (Over 3 h, up to 30 h)

Fluorescence anisotropy read LJL Acquest.

Definitions: Ki=dissociation constant for inhibitor binding

-   -   Kf=dissociation constant for fluorescent ligand binding

The fluorescent ligand is the following compound:

which is derived from5-[2-(4-aminomethylphenyl)-5-pyridin-4-yl-1H-imidazol-4-yl]-2-chlorophenoland rhodamine green.

Compounds of the invention have a K_(d) of less than 1 μM.

Raf Kinase Assay

Activity of human recombinant B-Raf protein was assessed in vitro byassay of the incorporation of radiolabelled phosphate to recombinant MAPkinase (MEK), a known physiologic substrate of B-Raf. Catalyticallyactive human recombinant B-Raf protein was obtained by purification fromsf9 insect cells infected with a human B-Raf recombinant baculovirusexpression vector. To ensure that all substrate phosphorylation resultedfrom B-Raf activity, a catalytically inactive form of MEK was utilised.This protein was purified from bacterial cells expression mutantinactive MEK as a fusion protein with glutathione-S-transferase(GST-kdMEK).

Method: Standard assay conditions of B-Raf catalytic activity utilised 3ug of GST-kdMEK, 10 uM ATP and 2 uCi ³³P-ATP, 50 mM MOPS, 0.1 mM EDTA,0.1M sucrose, 10 mM MgCl₂ plus 0.1% dimethylsulphoxide (containingcompound where appropriate) in a total reaction volume of 30 ul.Reactions were incubated at 25° C. for 90 minutes and reactionsterminated by addition of EDTA to a final concentration of 50 uM. 10 ulof reaction was spotted to P30 phosphocellulose paper and air dried.Following four washes in ice cold 10% trichloroacetic acid, 0.5%phosphoric acid, papers were air dried prior to addition of liquidscintillant and measurement of radioactvity in a scintillation counter.Results: The compounds of the examples were found to be effective ininhibiting B-Raf mediated phosphorylation of GST-kdMEK substrate havingIC₅₀'s of <3 μM.

The activity of compounds as Raf inhibitors may also be determined bythe assays described in WO 99/10325; McDonald, O. B., Chen, W. J.,Ellis, B., Hoffman, C., Overton, L., Rink, M., Smith, A., Marshall, C.J. and Wood, E. R. (1999) A scintillation proximity assay for theRaf/MEK/ERK kinase cascade: high throughput screening and identificationof selective enzyme inhibitors, Anal. Biochem. 268: 318-329 and AACRmeeting New Orleans 1998 Poster 3793.

The neuroprotective properties of B-Raf inhibitors may be determined bythe following in vitro assay:

Neuroprotective Properties of B-Raf Inhibitors in Rat Hippocampal SliceCultures

Organotypic cultures provide an intermediate between dissociatedneuronal cell cultures and in-vivo models of oxygen and glucosedeprivation (OGD). The majority of glial-neuronal interactions andneuronal circuitry are maintained in cultured hippocampal slices, sofacilitating investigation of the patterns of death among differing celltypes in a model that resembles the in vivo situation. These culturesallow the study of delayed cellular damage and death 24 hours, or more,post-insult and permit assessment of the consequences of long-termalterations in culture conditions. A number of laboratories havereported delayed neuronal damage in response to OGD in organotypiccultures of the hippocampus (Vornov et al., Stroke, 1994, 25, 57465;Newell et al., Brain Res., 1995, 676, 38-44). Several classes ofcompounds have been shown to protect in this model, including EAAantagonists (Strasser et al., Brain Res., 1995, 687, 167-174), Nachannel blockers (Tasker et al., J. Neurosci., 1992, 12, 98-4308) and Cachannel blockers (Pringle et al., Stroke, 1996, 7, 2124-2130). To date,relatively little is known of the roles of intracellular kinase mediatedsignalling pathways in neuronal cell death in this model.

Method: Organotypic hippocampal slice cultures were prepared using themethod of Stoppini et al., J. Neurosci. Methods, 1995, 37, 173-182.Briefly, 400 micron sections prepared from hippocampi of 7-8 daypostnatal Sprague Dawley rats are cultured on semiporous membranes for9-12 days. OGD is then induced by incubation in serum and glucose-freemedium in an anaerobic chamber for 45 minutes. Cultures are thenreturned to the air/CO₂ incubator for 23 hours before analysis.Propidium iodide (PI) is used as an indicator of cell death. PI is nontoxic to neurones and has been used in many studies to ascertain cellviability. In damaged neurons PI enters and binds to nucleic acids.Bound PI shows increased emission at 635 nm when excited at 540 nm. OnePI fluorescence image and one white light image are taken and theproportion of cell death analysed. The area of region CA1 is definedfrom the white light image and superimposed over the PI image. The PIsignal is thresholded and area of PI damage expressed as a percentage ofthe CA1 area. Correlation between PI fluorescence and histologicallyconfirmed cell death has been validated previously by Nissl-stainingusing cresyl fast violet (Newell et al., J. Neurosci., 1995, 15,7702-7711).

It is to be understood that the present invention covers all combinationof particular and preferred groups described herein above.

Throughout the specification and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer or step or group of integers but not to theexclusion of any other integer or step or group of integers or steps.

The application of which this description and claims forms part may beused as a basis for priority in respect of any subsequent application.The claims of such subsequent application may be directed to any featureor combination of features described herein. They may take the form ofcomposition, process, or use claims and may include by way of exampleand without limitation the following claims.

1. A compound of formula (I):

wherein: X is O, CH₂, S or NH, or the moiety X-R¹ is hydrogen; Y₁ and Y₂independently represent CH; R¹ _is hydrogen, C₁₋₆alkyl, C₃₋₇cycloalkyl,aryl, arylC₁₋₆alkyl, any of which except hydrogen is unsubstituted orsubstituted with substituent or substituents as defined below; R² and R³independently represent hydrogen, heterocyclyl, orheterocyclylC₁₋₆alkyl-, wherein the heterocycl or heterocyclylC₁₋₆alkylis morpholine; Ar is a group of the formula a) or b):

wherein A represents a fused 5 membered ring; R^(a)and R⁴ areindependently selected from hydrogen, halogen, C₁₋₆alkyl, aryl,arylC₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆ alkoxyC₁₋₆alkyl, haloC₁₋₆alkyl,arylC₁₋₆alkoxy, hydroxy, nitro, cyano, azido, amino, mono- anddi-N—C₁₋₆alkylamino, acylamino, arylcarbonylamino, acyloxy, carboxy,carbamoyl, mono- and di-N—C₁₋₆alkylcarbamoyl, C₁₋₆alkoxycarbonyl,aryloxycarbonyl, ureido, guanidino, C₁₋₆alkylguanidino, amidino,C₁₋₆alkylamidino, sulphonylamino, aminosulphonyl, C₁₋₆alkylthio,C₁₋₆alkyl sulphinyl or C₁₋₆alkylsulphonyl; and one of X₁ and X₂ is N andthe other is NR⁶, wherein R⁶ is hydrogen, C₁₋₆alkyl, or arylC₁₋₆alkyl;or a pharmaceutically acceptable salt thereof wherein the substituent orsubstituents for C₁₋₆alkyl or C₁₋₆cycloalkyl of R¹ as defined above isor are selected from: aryl, C₁₋₆alkoxy, C₁₋₆alkylthio, arylC₁₋₆alkoxy,aryl C₁₋₆alkylthio, amino, mono- or di-C₁₋₆alkylamino, aminosulphonyl,cycloalkyl, cycloalkenyl, COOH, amide, ureido, guanidino,C₁₋₆alkylguanidino, amidino, C₁₋₆alkylamidino, hydroxy, and halogen or acombination thereof; and wherein the substituent or substituents for anaryl, arylC₁₋₆alkyl, of R¹ as defined above is or are selected from:halogen, C₁₋₆alkyl, aryl, aryl C₁₋₆alkyl, C₁₋₆alkoxy, C₁₋₆alkoxyC₁₋₆alkyl, haloC₁₋₆alkyl, aryl C₁₋₆alkoxy, hydroxy, nitro, cyano, azido,amino, mono- and di-N—C₁₋₆alkylamino, acylamino, arylcarbonylamino,COOH, carbamoyl, mono- and di-N—C₁₋₆alkylcarbamoyl, C₁₋₆alkoxycarbonyl,aryloxycarbonyl, ureido, guanidino, C₁₋₆alkylguanidino, amidino,C₁₋₆alkyl amidino, sulphonylamino, aminosulphonyl, C₁₋₆alkylthio,C₁₋₆alkylsulphinyl, C₁₋₆alkylsulphonyl, or hydroxyimino-C₁₋₆alkyl or acombination thereof.
 2. A compound according to claim 1 wherein X—R¹ ishydrogen or X is N—H, provided that when X is N—H, R¹ is hydrogen orC₁₋₆-alkyl.
 3. A compound selected from:4-(1-Hydroxyimino-indan-5-yl)-5-pyridin-4-yl-1H-imidazole-2-carboxylicacid (2-morpholin-4-yl-ethyl)-amide; or a pharmaceutically acceptablesalt thereof.
 4. A pharmaceutical composition comprising a compoundaccording to claim 1 or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier.
 5. A pharmaceutical compositioncomprising a compound according to claim 3 or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier.